Hairpin ribozyme-antisense RNA constructs can act as molecular Lassos.
We have developed a novel class of antisense agents, RNA Lassos, which are capable of binding to and circularizing around complementary target RNAs. The RNA Lasso consists of a fixed sequence derived from the hairpin ribozyme and an antisense segment whose size and sequence can be varied to base pair with accessible sites in the target RNA. The ribozyme catalyzes self-processing of the 5'- and 3'-ends of a transcribed Lasso precursor and ligates the processed ends to produce a circular RNA. The circular and linear forms of the self-processed Lasso coexist in an equilibrium that is dependent on both the Lasso sequence and the solution conditions. Lassos form strong, noncovalent complexes with linear target RNAs and form true topological linkages with circular targets. Lasso complexes with linear RNA targets were detected by denaturing gel electrophoresis and were found to be more stable than ordinary RNA duplexes. We show that expression of a fusion mRNA consisting of a sequence from the murine tumor necrosis factor-alpha (TNF-alpha) gene linked to luciferase reporter can be specifically and efficiently blocked by an anti-TNF Lasso. We also show in cell culture experiments that Lassos directed against Fas pre-mRNA were able to induce a change in alternative splicing patterns.
Dallas
Anne
A
SomaGenics, Inc, Santa Cruz, CA 95060, USA.
Balatskaya
Svetlana V
SV
Kuo
Tai-Chih
TC
Ilves
Heini
H
Vlassov
Alexander V
AV
Kaspar
Roger L
RL
Kisich
Kevin O
KO
Kazakov
Sergei A
SA
Johnston
Brian H
BH
D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D005786:Gene Expression Regulation; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012313:RNA / Q000737:chemistry; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D016372:RNA, Antisense / Q000737:chemistry* / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D000079962:RNA, Circular; D012333:RNA, Messenger / Q000737:chemistry / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics; D019014:fas Receptor / Q000235:genetics
D012322:RNA Precursors; D016372:RNA, Antisense; D016337:RNA, Catalytic; D000079962:RNA, Circular; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D019014:fas Receptor; C418481:hairpin ribozyme; D012313:RNA